This application claims priority from PCT/FR98/02699 filed Dec. 11, 1998, and from French patent application 97/15702 filed Dec. 11, 1997.
The present invention relates to dextran derivatives, and to their applications as medicines with specific biological action, such as a cicatrizing action, an anti-complement action (substitute for plasma), a proliferation modulating action or an anticoagulant action, and more specifically an anti-thrombotic action, as well as to a process for their preparation.
Various dextrans substituted with side chains bearing carboxylate and sulfonate groups have been described. In particular, French patent 2,461,724 and French patent 2,555,589 describe dextrans substituted with said groups, which respectively display anticoagulant properties and anticoagulant and anti-inflammatory properties; European patent 0,402,194 describes the cell and tissue regeneration properties of such substituted dextrans.
It has also been shown that such substituted dextrans can have other biological activities, depending on the degree of substitution with said groups; in particular, European patent 0,514,449 describes dextrans (D) substituted with carboxymethyl (CM) and carboxymethylbenzylamide sulfonate (BS) groups of general formula DXCMYBSZ, in which X, which represents the average number of unsubstituted saccharide units per 100 saccharide units, is less than or equal to 50, Y, which represents the average number of carboxymethyl groups per 100 saccharide units, is between 10 and 90, and Z, which represents the average number of carboxymethylbenzylamide sulfonate groups per 100 saccharide units, is between 15 and 35; to give an agent for inhibiting the growth of tumor cells.
These various derivatives, the structure of which is summarized in FIG. 1, are generally obtained by random substitution of dextran with three different groups: carboxymethyl (CM), carboxymethylbenzylamide (B) and carboxymethylbenzylamide sulfonate (S) (sulfonation on the aromatic ring with chlorosulfonic acid).
More specifically:
a) the carboxymethylation of dextran (production of CMD) is carried out in basic aqueous medium, by the action of monochloroacetic acid. Three successive carboxymethylation reactions are required to obtain a degree of substitution (ds) of the dextran, expressed relative to the number of free hydroxyl functions in a glucoside unit of the dextran, of between 0.7 and 1.1;
b) the coupling of benzylamine to the carboxymethyl groups (production of CMDB) is based on the ability of the carboxylate function to form an unstable mixed anhydride capable of reacting with a reagent bearing a primary amine function (Rxe2x80x94NH2). Two different processes or activation reactions were used to achieve the formation of a mixed anhydride:
action of isobutyl chloroformate (IBC) or
action of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ).
In both cases, the reaction is carried out in a heterogeneous medium: water/dimethylformamide or water/ethanol, respectively.
These two coupling processes give similar degrees of substitution (ds), of about 0.08 to 0.12, in a single step. In the case of coupling with EEDQ, the ds can reach 0.25 to 0.30, in a single step, when the intermediate product is activated at a temperature of 30 to 40xc2x0 C.
As with the carboxymethylation, to achieve high ds values for benzylamine, it is not possible to increase the concentrations of the various reagents. It is thus necessary to perform successive couplings in order to improve the reaction yield. The CMDB precipitated, washed and dried after the first coupling undergoes a second and/or a third coupling under exactly the same conditions as the first, without consideration of the substitutions due to the first coupling.
c) the sulfonation or sulfation takes place by the action of monochlorosulfonic acid on the CMDB, in anhydrous organic medium (for example dichloromethane) and in heterogeneous phase, the CMDB not being soluble in dichloromethane. In such a heterogeneous medium, the distribution of the sulfates in the saccharide units occurs unequally (Ricketts, C. R., J. Chem. Soc., 1956, 3752-3756). It is necessary to perform the reaction in excess monochlorosulfonic acid, while at the same time avoiding acid hydrolysis of the polysaccharide chain.
The [HSO3Cl]/[bound B units] ratios for the CMDBs and [HSO3Cl]/[free OH] ratios for the CMD range between 0.8 and 3.
In order essentially to obtain a sulfonation of the aromatic rings (B units), the [HSO3Cl]/[bound B units] molar ratio should be equal to 3, whereas, in order to obtain a sulfation of the hydroxyl functions borne by the glucoside units (production of sulfate functions), the [HSO3Cl]/[free OH] molar ratio is about 1. In any case, the concentration of chlorosulfonic acid in the reaction medium should not exceed 0.15 M. Under these conditions, the percentage of units bearing a sulfonate function depends on the percentage of units substituted with benzylamide groups. A recent study (Maiga-Revel O. et al., Carbohydrates Polymers, 1997, 32, 89-93) has shown that the anticoagulant activity of the CMDSu (=carboxymethyldextran sulfate) and CMDBS (=carboxymethyldextran benzylamide sulfonate) dextran derivatives of the prior art depends on their sulfur content; however, CMDBSs have better anticoagulant activity than that obtained with CMDSu""s, for an identical sulfur content.
The dextran derivatives obtained under the conditions defined above have the drawback of having irregular distribution of the chemical groups and of the sizes of the polysaccharide chains, leading to a heterogeneous final product whose properties are difficult to control.
The inventors have developed a novel process for preparing dextran derivatives, which allows better control of the production of specifically defined products (controllable degree of substitution, homogeneity of the distribution of the charged or uncharged chemical groups and size homogeneity of the polysaccharide chains in the final product, selection and better reproducibility of the desired activity).
A subject of the present invention is dextran derivatives of general formula DMCaBbSucSd, in which:
D represents a polysaccharide chain, preferably consisting of concatenations of glucoside units,
MC represents methylcarboxylate groups,
B represents carboxymethylbenzylamide groups,
Su represents sulfate groups (sulfation of the-free hydroxyl functions borne by the glucoside units),
S represents sulfonate groups (sulfonation of the aromatic rings),
a, b, c and d represent the degree of substitution (ds), expressed relative to the number of free hydroxyl functions in a dextran glucoside unit, with groups MC, B, Su and S, respectively; a being equal to 0 or xe2x89xa70.3, b being equal to 0 or xe2x89xa70.1, c being equal to 0 or xe2x89xa70.1 and d being equal to 0 or xe2x89xa70.15, with the proviso that when d=0, a and/or b are xe2x89xa00,
which products display:
homogeneity of the size distribution of the chains, illustrated by an elution profile of symmetrical Gaussian type in high performance steric exclusion chromatography, and
homogeneity of the distribution of charged chemical groups, illustrated by an elution profile as a single symmetrical peak in low-pressure ion exchange chromatography.
These products are considered as being copolymers consisting of fictive subunits Rxe2x80x94OH and Rxe2x80x94OX, it being possible for X to be a methylcarboxylate (MC) benzylamide (B), sulfate (Su) or sulfonate (S) group, the polysaccharide chain of the unsubstituted dextran being considered as consisting of 300 fictive Rxe2x80x94OH subunits, instead of 100 glucoside units, with regard to the fact that an unsubstituted glucoside unit comprises three free hydroxyl groups. Thus, a dextran methylcarboxylate (DMC) with a degree of substitution (ds) with methylcarboxylate groups of 1.2 contains 1.20 substituted groups (Rxe2x80x94MC) and 1.80 free hydroxyl groups (Rxe2x80x94OH), per glucoside unit.
This thus gives, in contrast with the heterogeneous products of the prior art, homogeneous products, of targeted composition, in which the bioactive chemical groups are distributed along the macro-molecular chains in a specific order, giving the product a biological property which will not be found in a product of the same overall composition but in which the distribution of said groups along the macromolecular chains is different (different preparation, in particular).
In other words, in the dextran derivatives according to the invention, the distribution of the chemical groups gives the final product a specific biological property; the consequence of such a distribution is that the chemical composition of each polysaccharide chain is identical to the overall chemical composition of the product. Accordingly, there is an optimum chemical composition for a maximum specific biological activity; there is thus a direct relationship between the biological property considered and the overall chemical composition of the product.
For example:
the dextran derivatives of general formula DMCaBbSucSd as defined above, in which axe2x89xa70.6, bxe2x89xa00, c equal to 0 or xe2x89xa60.5 and dxe2x89xa60.15 or d equal to 0 are essentially cicatrizing agents, preferably when they have a molar mass between 3000 and 500,000 g/mol; the dextran derivatives preferred as cicatrizing agents are those in which a is between 0.7 and 0.9; cxe2x89xa60.5 and dxe2x89xa60.15 or equal to 0;
the dextran derivatives of general formula DMCaBbSucSd as defined above, in which axe2x89xa70.3, bxe2x89xa00, c equal to 0 or xe2x89xa60.4 and dxe2x89xa60.15 or equal to 0 are essentially agents with anti-complement activity and plasma substitutes, preferably when they have a molar mass of between 10,000 and 60,000 g/mol; the dextran derivatives preferred as agents with anti-complement activity and as substitutes for plasma are those in which a is between 0.40 and 1.15, bxe2x89xa60.4, cxe2x89xa60.2 and dxe2x89xa60.15 or equal to 0;
the dextran derivatives of general formula DMCaBbSucSd as defined above, in which axe2x89xa70.5, bxe2x89xa00, c equal to 0 or xe2x89xa60.4 and dxe2x89xa60.15 are essentially agents for modifying cell proliferation, preferably when they have a molar mass of between 3000 and 100,000 g/mol; the dextran derivatives preferred as cell proliferation modulating agents are those in which a is between 0.5 and 1.2; b is between 0.2 and 0.6; c is between 0.1 and 0.4 and dxe2x89xa60.15 or equal to 0; and
the dextran derivatives of general formula DMCaBbSucSd as defined above, in which axe2x89xa70.4, cxe2x89xa70.3 and dxe2x89xa60.15 or equal to 0 are essentially anticoagulant agents, preferably when they have a molar mass of between 3000 and 20,000 g/mol and a value for bxe2x89xa00.
A subject of the present invention is also medicines, characterized in that they comprise as active principle at least one dextran derivative as defined above, optionally combined with another active principle and/or with at least one pharmaceutically acceptable vehicle and/or a physiologically acceptable support, preferably a liposome.
The combined active principles are chosen from the group comprising dextrans, growth factors (for example an acidic fibroblast growth factor (FGF) or a basic FGF), local anesthetics, anti-infection agents, seric proteins and collagen.
A subject of the present invention is also a process (process 1) for preparing dextran derivatives of general formula DMCaBbSucSd, as defined above, characterized in that it comprises the following steps:
a) carboxymethylation comprising (i) activation of an unsubstituted dextran, by placing said dextran in contact with a basic two-phase liquid aqueous-alcoholic medium for at least 1 h with stirring, (ii) addition of monochloroacetic acid to the activated product obtained, at a temperature of between 40 and 90xc2x0 C., preferably at 60xc2x0 C., the ratio RMC, equal to the number of moles of monochloroacetic acid/number of moles of OH, being between 0.3 and 2, (iii) isolation and optionally purification of the dextran methylcarboxylate (DMC) obtained.
b) coupling of benzylamine with methylcarboxylate groups (benzylamidation) comprising (i) the placing in contact, for at least 2 h and in an acidic aqueous medium, of the DMC obtained in a) with a primary amine (benzylamine), in the presence of a water-soluble carbodiimide such as 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide meta-p-toluene sulfonate (CMC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) as coupling agent, at a temperature of between 0xc2x0 C. and 30xc2x0 C., the CMC/MC molar ratio being between 0.25 and 2 and the benzylamine/MC molar ratio being between 0.25 and 2, (ii) isolation of the dextran methylcarboxyl benzylamide (DMCB) obtained and optionally purification thereof.
Such a step, performed in homogeneous medium and in the presence of a water-soluble carbodiimide as coupling reagent, allows better control of the reaction and thus preparation of the final product, this product having a homogeneity of the chain size distribution, illustrated by an elution profile of symmetrical Gaussian type in high performance steric exclusion chromatography and a homogeneity of the distribution of charged chemical groups, illustrated by an elution profile as a single symmetrical peak in low-pressure ion exchange chromatography.
c) sulfation comprising (i) the formation of a trialkylammonium salt of the DMCB obtained in b), (ii) solubilization of the salt obtained in an anhydrous polar solvent, generally a Lewis base (electron donor), such as dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) and (iii) addition, to said dissolved salt, of a complex based on sulfur trioxide such as SO3-pyridine, SO3-triethylamine or SO3-DMF dissolved in the same solvent, at a temperature of less than 70xc2x0 C., the complex based on sulfur trioxide/free OH molar ratio being between 0.25 and 12, and optionally
d) sulfonation of the groups B by mixing, with stirring, a derivative DMCBSu in suspension in an anhydrous solvent with chlorosulfonic acid dissolved in the same solvent, at a temperature between room temperature and the boiling point of the solvent used.
Unexpectedly, the process according to the invention allows the degree of substitution of the dextran to be controlled, in a number of steps which is significantly smaller than the number of steps in the processes of the prior art, to give products with an elution profile of symmetrical Gaussian type in high performance steric exclusion chromatography and an elution profile as a single symmetrical peak in low-pressure ion-exchange chromatography and the desired biological activity, with yields which are significantly higher than those of the prior art.
In addition, the process according to the invention makes it possible to reproducibly synthesize a product of desired chemical composition for the biological property selected. Thus, a biological activity preferably corresponds to a given chemical composition of a product.
For example, step a) gives, in a single step, a ds with MC of 1.0xc2x10.1 per glucoside unit and a yield of greater than or equal to 80%, for an RMC value=0.85, in a water/tert-butanol or water/isopropanol (15/85 v/v) mixed medium, with stirring for 2 hours at 60xc2x0 C.; starting with a DMC with a ds with MC=1, for a CMC/MC molar ratio of 0.75 and a benzylamine/MC molar ratio of 1, and with stirring at room temperature for 16 hours, step b) gives a DMCB product which has a ds with MC of 0.70xc2x10.05 and a ds with B of 0.30xc2x10.03, with a yield of greater than 80%; and step c) gives a yield of greater than or equal to 60%.
According to one advantageous embodiment of said process, in step a), the water/alcohol ratio is between 10/90 (v/v) and 25/75 (v/v) and is preferably 15/85 (v/v).
As a variant, the process (process 2) for preparing the dextran derivatives of general formula DMCaBbSucSd, as defined above, in which a=0 or axe2x89xa00, b is other than 0 and d=0, comprises the following steps:
a) preparation of N-methylphenyl-2-chloroacetamide by placing a benzylamine in contact with chloroacetyl chloride, followed by isolation and purification of the product,
b) placing dextran, dissolved in basic aqueous-alcoholic solution, in contact successively with N-methylphenyl-2-chloroacetamide in alcoholic solution obtained in a) in the presence or absence of monochloroacetic acid in alcoholic solution, maintaining the mixture obtained at a temperature above 40xc2x0 C., preferably at 60xc2x0 C., followed by isolation and optionally purification of the DB or DMCB obtained, and
c) sulfation of the product obtained in b) comprising (i) formation of a trialkylammonium salt, (ii) dissolution of the salt obtained in an anhydrous polar solvent, generally a Lewis base, such as dimethyl sulfoxide (DMSO) or dimethylformamide (DMF), (iii) addition, to said dissolved salt, of a complex based on sulfur trioxide such as SO3-pyridine, SO3-triethylamine or SO3-DMF dissolved in the same solvent, at a temperature below 70xc2x0 C., the complex based on sulfur trioxide/free OH molar ratio being between 0.25 and 12, and (iv) isolation and optionally purification of the DBSu or DMCBSu obtained.
The presence of steps b) and c) above makes it possible to obtain the same homogeneity of the final product as that obtained with process 1 above.
In order to prepare the dextran derivatives of general formula DMCaBbSucSd, as defined above, in which a is other than 0, b=0 and d=0 (xe2x86x92DMCSu) (process 3), said process is characterized in that it comprises the following steps:
a) carboxymethylation of an unsubstituted dextran, under the conditions outlined above, and
b) sulfation of the DMC obtained in a) under the conditions outlined above.